Protocol for the induction of innate immune memory in human smooth muscle cells and endothelial cells in vitro

Summary Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2

Note: The amount of serum may vary depending on the cell type, for example, if using human umbilical vein endothelial cells (HUVECs), the amount of serum can be reduced to 2%, whereas human aortic endothelial cells (HAECs) require 5% serum.
Alternatives: Replace the Human Vascular Smooth Muscle Cell Basal Medium with Smooth Muscle Cell Basal Medium 2 from PromoCell.
Alternatives: Replace the RPMI-1640 Medium with any other basal buffered medium such as DMEM.

STEP-BY-STEP METHOD DETAILS
Pre-coating the culture flask or plates for HUVECs/HAECs Timing: 1 h 1x Phosphate-buffered saline (PBS).
1% gelatin solution in sterile filtered water.
1.If using HAECs/HUVECs, the cell culture vessels need to be pre-coated with 1% gelatin (problem 1): a. Add 4 mL 1% gelatin solution to T75 cell culture flasks, 1 mL or 0.5 mL per well on 6 well plates or 12-well plates, respectively.b.Carefully spread the gelatin by moving the flask or plate in a cross-like pattern without creating bubbles until the entire bottom is coated with gelatin.c.Remove (z3.5 mL) excess gelatin without leaving any bubbles.d.Put the culture flask and plate at 37 C for 30 min.e. Remove the excess gelatin once again (as much as possible without altering the coating (About few hundred microliters)) and leave the vessels under the hood for complete drying (for 30 min).f.Rinse the vessels with 1 mL or 0.5 mL of sterile PBS per well for 6 well plates or 12-well plates respectively before adding cell suspension.
Note: If the cell culture flasks are closed airtight and sealed with parafilm, they can be stored at 4 C for a maximum of 3 days.
Note: When using a suction pump, carefully remove the gelatin excess without touching the bottom.
2. Calculate the needed culture surface area according to the plating density (see Table 1).Prewarm any needed media for the planned step in a 37 C water bath including: a. VSMCs: i. Smooth muscle cell growth medium (SMGM).b.HAECs/HUVECs: i. Endothelial cell growth medium (ECGM).c. 1x Phosphate-buffered saline (PBS).d. 1% gelatin solution.
3. Label the cell culture flasks and plates with the cell type, the passage and date.

Timing: 45 min
Note: Cryopreserved cells should be stored at -196 C in a liquid nitrogen tank.Short-or longterm storage at -20 C will result in a severe decrease in cell viability, and long-term storage at -80 C is not sufficient for cell preservation.4. Add 13 mL SMGM or ECGM to a 75 cm 2 cell culture flask or a precoated 75 cm 2 cell culture flask for culturing SMCs or HAECs/HUVECs respectively.Allow the culture vessel to warm up and equilibrate in an incubator for at least 30 min. 5. Take a cryovial containing the cells from the liquid nitrogen tank and immediately put it into a 37 C water bath for 2 min.
Note: For safety reasons, when removing the cryovial from the liquid nitrogen tank, twist the cap partially open to relieve pressure and then retighten.Omitting this step may cause over pressurization of the cryovial during thawing, resulting in an explosion, body damage, and cell lost.
6. Wipe the cryovial and disinfect it with 70% Ethanol before opening it in a sterile bench.7. Resuspend the cells by gently pipetting with a 1000 mL pipette.8. Dispense the cells carefully in the respective prewarmed 75 cm 2 cell culture flask, loosely tighten the cap.
Note: Make sure that the cap for the flask is designed for sterile air exchanges.9. Gently move the flask in cross-like pattern to distribute the cells homogenously.10.Return the flask to the humidified 37 C incubator with 5% CO 2 .11. Refresh the SMGM or ECGM the following day to remove the dimethyl sulfoxide (DMSO) residue in the freezing medium and any unattached cells.
Alternatives: Transfer the cryovial content to a 15 mL tube and increase the volume with PBS or medium and centrifuge at 350 g for 5 min.Discard the freezing medium containing DMSO.
Resuspend the cells in the respective medium prior to adding to the cell culture flask.This allows skipping step 8 but causes some cell loss due to the centrifugation.However, based on experience, the cells show a better performance when they are seeded directly.
12. Replace the medium after every two to three days until the confluency reaches up to 80%-90% for subcultivation.
Note: DMSO in a high concentration can have impact on cell growth and viability.

Subcultivation of vascular cells
Timing: 1.5 h Before starting.
Prepare the reagents and prewarm at 37 C.
Detachment stop medium.
Note: Following thawing of the cryopreserved cells, proliferation might be delayed for 1-2 days (lag-phase).Afterwards, the cells will begin to proliferate logarithmically (log-phase).Due to their phenotypic plasticity, it is mandatory to avoid over confluency.The cells should be kept in the log-phase, and subcultivation should be considered when reaching about 70%-80% confluence (Figures 1A, 1B, 2A, and 2B).
13. Remove the cell culture medium and carefully rinse the cells twice with 5 mL of prewarmed PBS, swirling the liquid around inside the cell culture flask or plate before aspirating and discarding it.14. Add 5 mL Detachment solution per 75 cm 2 cell culture flask and place it into the incubator for 2-3 min.15.Examine the cells under microscope and control cell detachment regularly.When the cells start detaching, slowly tap the side of the flask to loosen the remaining cells (problem 2).16.Immediately add 5 mL Detachment stop medium.
Note: FBS in the Detachment stop medium is essential for neutralizing the effect of Accutase or Trypsin.17. Transfer the cell suspension to a new 50 mL falcon tube.18. Wash out the remaining cells by adding 5 mL PBS and rinsing in all areas of the flask.19.Resuspend the cells to make a homogenized suspension.20.Count the cells in a Neubauer chamber under a microscope or using another available cell counter device.
21. Centrifuge the tube at 350 g for 8 min at room temperature.22. Aspirate and discard the supernatant and resuspend the cell pellet in 1 mL of appropriate growth medium (problem 2).23.Calculate required number of the cells and make a cell suspension with desired cell concentration and amount of SMGM or ECGM, according to the recommended seeding density (Table 1).

Figure 1. Morphological observation and identification of HUVECs
Light microscopy of HUVEC density 24 h after seeding (A) and when reaching about 80% density (B).HUVEC were cultured in EBM-2 growth medium.In 1% gelatin coated vessels.The photos were taken under light microscopy with 4x magnification object.

Protocol
Note: Carefully resuspend the cells by gently pipetting up and down to make a single cell suspension.
Note: The number of cells for seeding can be adjusted based on the proliferative abilities and thereby the time to 80% confluence of the cell type and according to donor variability.
24. Gently move the plate or flask in a small cross-like pattern to distribute the cells evenly (problem 2).
Note: Check the cells under a microscope, and make sure that cells are distributed equally across the well.Uneven cell distribution may have a big impact on the experiment results.
25. Place the cell culture plates in an incubator and change the media every two to three days.
26. Check the cells under the microscope, assuring a lack of contamination.If the confluency is approximately 70%, proceed to the next step.27.Change the cell culture medium to resting medium for a 24 h starvation period prior to priming (1 mL per well on a 12-well plate).a. Discard the old medium using a suction pump or pipette.
CRITICAL: Avoid touching the cells with pipette tips or letting them dry out.If using a suction pump, avoid disrupting the cell layer by sucking the well completely dry.
b. Add fresh medium by pressing the tip of the pipette against the wall and letting it run in slowly to avoid mechanical cell damage.
CRITICAL: High pipetting pressure can damage the cell layer.Gently add the medium and avoid disrupting the cells.
28.When using pharmacological inhibitors in an experiment, add them at least 1 h prior to adding the priming reagent (problem 4).
CRITICAL: When using an organic solvent such as DMSO, prepare corresponding controls.
29. Add the required dilution of the priming reagent (e.g., 10 mg/mL oxLDL), if necessary dilute in SMRM or ECRM.Shake the cell culture plate carefully to ensure equal distribution of oxLDL and return it to the incubator.30.After 24 h change the cell culture medium to fresh resting medium, thereby ending the priming and starting the resting time.

Timing: 4 days
The vascular cells are incubated in resting medium for four days, before harvesting them for subsequent analysis or restimulating them with a secondary unspecific inflammatory stimulus.
31.Refresh the SMRM or ECRM after every two or three days (problem 5).CRITICAL: It is very important to find an appropriate dilution of the samples per well.This can vary and depend on the cell type and cell culture condition.For instance, for detecting IL-8 concentration after Pam3Cys4 restimulation in SMCs, it is necessary to dilute the sample between 20-50 times.
Note: Incubation of the plates containing the samples can be optimized.For detecting IL-1b, VEGF1, or other targets with low concentration, we recommend to incubate the plates containing supernatant overnight at 4 C.
42. Adding the detection antibody: a. Prepare the required volume of detection (secondary) antibody.The concentration of the detection antibodies for IL-6 and IL-8 are 50 and 20 ng/mL respectively.
CRITICAL: Briefly centrifuge the antibody before use.
b. Flip the plates and wash 3x with 150 mL/well washing buffer.c.Add an appropriate volume of the detection antibody/well.Seal the plate and incubate 2 h at room temperature on a shaker.43.Adding Avidin-HRP (-horseradish peroxidase): a. Prepare required volume of Avidin-HRP according to the manufacturers guidelines.b.Flip the plates and wash 3x with 150 mL/well washing buffer.c.Blot the plate on a paper towel to remove any residual buffer.
d. Add 100 mL/well Avidin-HRP to all wells.Put the plate in the dark with a lid on and incubate at room temperature for 20-30 min.
CRITICAL: Avidin-HRP must be kept in the dark, avoid incubating the solution or the plates in the light.
44. Adding HRP substrate ( : a. Prepare required volume of HRP substrate.b.Wash at least 4x with 150 mL/well washing buffer.c.Add 100 mL substrate/well.Incubate the plates at room temperature for 10-20 min in the dark.
Note: Check the plates repeatedly in order to prevent saturation of color development.
45.To terminate the HRP/TMB reaction, add 50 mL of the stop solution into the wells.
Caution: Wear protective gloves, clothing, and eye and face protection.
46. Measure the color absorbance at 450 nm with a plate reader.

EXPECTED OUTCOMES
The goal of this protocol is, to induce and analyze an innate immune memory in vascular cells.Therefore, the expected result is an enhanced immune response in primed VSMCs and HAECs/HUVECs upon secondary stimulation such as Pam3Cys4 (Figures 4, 5, and 6A-6D).This is represented in terms of increased concentrations of inflammatory cytokines such as IL-6 and IL-8 in comparison to a control group lacking the primary stimulus.These can be measured via ELISA or real-time qPCR.

QUANTIFICATION AND STATISTICAL ANALYSIS
For processing raw ELISA data, any software, which creates four parameters logistic regressions such as WorkOut 2.5 by Dazdaq or the MyAssays online program can be used (Figure 7).

Note:
The response and level of cytokines may differ among the donors.Basic level of IL-6 and IL-8 usually is higher in vascular cells in comparison to monocytes.

LIMITATIONS
This is an in vitro approach for the induction of trained immunity and may not reflect the complexity of in vivo experiments.The protocol is optimized for human vascular cells using limited donors.It is applicable to other cell types and donors.However, it may need further adjustments and optimization regarding timing and concentrations of chemicals.

TROUBLESHOOTING Problem 1
No coating or partial coating of 1% gelatin.

Potential solution
When coating the plates with 1% gelatin, creation of bubbles or solid particles may cause uneven coating and results in not homogenous confluence of the cells.Slowly shake the flasks or the plates.Make sure that gelatin solution covers the surface area completely.Make sure that no solid particles or bubbles are left after the coating.Monitor the coating quality by slightly shaking the flask or plate.Pipet out any solid particles and the excess gelatin solution over the bubble until it has disappeared.

Problem 2
Suboptimal cell distribution after sub-cultivation of VSMCs or endothelial cells.

Potential solution
When adding the Detachment solution, any serum left in the well/flask might inactivate the solution and lead to uneven detachment results.
In addition, leaving cells in Detachment solution for too long when subcultivating, may damage them and lead to cell clumping.Fast detachment of the cells may lead to cell damage.Moreover, the same might occur to already detached cells if the overall process takes > 5 min.Reduce or increase the amount of the Detachment solution used, to detach the cells accordingly.Remember to adjust the amount of detachment stop medium as well and control multiple times for cell detachment, in order to stop the reaction, before causing severe damage to the cells.
After centrifugation, resuspend the cells in less medium.Clumping can be prevented by pipetting a little more vigorously and repeatedly, yet this may result in cell damage.Furthermore, when distributing cells mechanically in the wells after seeding, less movement is sometimes better.Moving plates in circular patterns may lead to uneven distribution of the cells.Move your well plates slowly and carefully after seeding, e.g., when putting them into the incubator and assure that the shelves in the incubator are level, if experiencing a drift of cells towards one side of the wells.

Potential solution
Make sure that the Detachment solution is prewarmed to 37 C, as the detachment process is not efficient when using cooled solutions.
Any serum left in the flask might inactivate the Accutase contained in the Detachment solution.Always wash at least two times with PBS before adding the Detachment solution.If there are cells left in the flask after the detachment, the cycle of adding Detachment solution and stopping the reaction can be repeated.Rather repeat the cycle than incubating the cells longer with Accutase, as this might damage the already detached cells.If doing so, remember to wash the flask again with PBS before adding the Detachment solution, to minimize serum left from the Detachment stop medium.

Problem 4
Data in between multiple runs is deviating.

Potential solution
This might be due to basic donor variation if using multiple cell lines.Cells may also behave differently, when used in higher passages.For comparability, we recommend to use the cells up to passage 7. Another reason may be pipetting mistakes.
Firstly, check if the pipette tip is attached tightly.Differently attached tips might lead to different volumes pipetted every time the tip is changed, e.g., when air is aspirated because the tip is not firmly attached.Be extremely aware of this issue, when using a multichannel pipette.
Secondly, avoid pipetting small volumes.When pipetting volumes < 2 mL, small variations and droplets stuck to the tip can affect heavily on the results.Therefore, dilute smaller volumes in cell culture medium before adding to the wells.
Lastly, most pipette tips feature scale ring marks according to different volumes, which facilitate the control of the aspirated volume.
Bacterial or fungal contamination may lead to imprecise results.Therefore, regularly scan your well plates with an optical microscope, using magnifications such as 10x or 20x to spot any contamination.

Problem 5
Cell detachment and cell death after adding chemicals to the cell culture.

Figure 2 .
Figure 2. Morphological observation and identification of VSMCs Light microscopy of VSMC density 24 h after seeding (A) and when reaching about 70% density (B).VSMCs were cultured in M231 medium supplemented with SMCs growth supplement.The photos were taken under light microscopy with 4x (A) and 10x (B) magnification object.

Figure 3 .
Figure 3. Representation of the presented protocol for induction of trained immunity in vascular cells Timeline in days.

Figure 4 .
Figure 4. Expected results of inducing innate immune memory The primed cells are expected to show enhanced immune reaction in response to a secondary stimulus.

Figure 5 . 3
Figure 5. Example of a standard curve for analyzing ELISA data

Figure 6 .
Figure 6.oxLDL priming induces a proinflammatory phenotype in HUVECs and VSMCs oxLDL induced trained immunity in VSMCs (A, B) and HUVECs (C, D).VSMCs and HUVECs were primed with 20 mg/mL of oxLDL for 24 h.The medium was changed and the cells were rested for 4 days.Then, the cells were restimulated with 5 mg/mL of Pam3Cys4 for 24 h and IL-6 and IL-8 concentration were measured using ELISA.Graphs represent mean values G SD. *p < 0.05.

Figure 7 .
Figure 7. oxLDL priming induces upregulation of inflammatory genes in HUVECs and VSMCs oxLDL induced innate immune memory in VSMCs (A, B) and HUVECs (C, D).VSMCs and HUVECs were primed with 20 mg/mL of oxLDL for 24 h.The medium was changed and the cells were rested for 4 days.Then, the cells were restimulated with 5 mg/mL of Pam3Cys4 for 6 and lysed for RNA isolation.IL-6 and IL-8 mRNA expression were measured using qPCR.Graphs represent mean values G SD. *p < 0.05.
Replace the Human Vascular Smooth Muscle Cell Basal Medium and Smooth Muscle Growth Supplement with Smooth Muscle Cell Growth Medium 2 (# C-22062) from PromoCell GmbH.
Store at 4 C; protect from light; do not freeze; stable until expiration date.Alternatives:

Table 1 .
Medium requirements Stop solution: 1 M Hydrochloric acid (HCl) or 1 M Sulfuric acid (H 2 SO 4 ). .Coat the ELISA plate with suggested volume/well of the coating buffer.c.Seal and incubate the plate overnight at room temperature.Discard the coating solution and wash the plate 3x with 150 mL/well washing buffer.b.Tap the plate on a paper towel to remove any residual buffer.c.Block the wells with 150 mL/well 1x Reagent Diluent and incubate for at least 1 h at room temperature.41.Preparing the standards and adding the samples: ba. Prepare the standard in duplicates in a serial dilution (11 standards + blank).b.Wash 3x with 150 mL/well washing buffer.The concentration of the highest standard for IL-6and IL-8 are 1200 and 4000 pg/mL respectively.Note: Do not let the wells dry up completely.c.Add the required dilution of the standards in the wells.d.Add proper volume of the samples with appropriate dilution (diluted in assay diluent) to the well.Seal the plate and incubate at room temperature for 2 h on the shaker.